Immunoglobulin conjugates

ABSTRACT

Immunoglobulin conjugates formed by reacting a difluoronucleoside with an immunoglobulin via an alkane dioic acid linking group.

CROSS-REFERENCE

This application is a division of application Ser. No. 07/124,191, filedNov. 23, 1987, now U.S. Pat. No. 4,814,438, which is acontinuation-in-part of copending application Ser. No. 06/946,351, filedDec. 24, 1986.

BACKGROUND OF THE INVENTION

Despite the development of numerous chemical agents and sophisticatedregimens of drug therapy, the ravages of cancer continue to extract anever-increasing human toll of suffering and death. Although manyadvances have been made, especially in the area of combination drugtherapy, the need for new and better methods of treating neoplasms andleukemias has not diminished. This is especially evident in the area ofinoperable or metastatic solid tumors, such as various forms of lungcancer.

While the treatment of cancer was once considered impossible, greatstrides have been made during the past ten years in controlling theravages of this often fatal disease. Several drugs which contribute tothe increasing rate of survival are now routinely used clinically. Themost commonly employed antitumor agents include methotrexate,doxorubicin and vinca alkaloids such as vincristine. However, researchcontinues in an attempt to develop more effective compounds with greatersafety. This invention provides valuable improvements in the treatmentof tumors.

EPO Patent Application Publication No. 184,365 describes the use ofcertain difluoronucleosides for the treatment of neoplasms in mammals.The antiviral use of some of these same nucleosides and methods fortheir preparation are disclosed in U.S. Pat. No. 4,526,988 and BritishPatent Application No. GB 2172287. These compounds were found to haveuseful activity against a variety of tumor systems in mice.

The present invention provides immunoglobulin conjugates of some ofthese difluoronucleosides. Although the general concept of conjugatingdrugs to antibodies is generally known, e.g., EPO Patent Application No.88695, the literature clearly acknowledges the manner and means forwhich conjugation is accomplished can be critical to obtainingconjugates having useful biological properties. For example, where acompound or drug has more than one reactive functional group, attachmentthrough one functionality may provide a conjugate with significantlygreater or lesser biological activity as compared with conjugationthrough a different functional group. Similarly, the manner in whichsuch conjugation occurs is often critical to the biological utility. Insome instances, better biological activity is obtained by covalentlylinking the compound and antibody directly whereas in other cases alinking group of some type between the two moieties is preferred. Thus,in most cases, there is no way of predicting whether a particular mannerof conjugation will provide a useful conjugate.

SUMMARY OF THE INVENTION

The present invention provides immunoglobulin conjugates of the generalformula (Q)_(n) -Im where Im is an immunoglobulin or immunoglobulinfragment, n is about 1-10, and Q is an acylated difluoronucleoside ofthe formula ##STR1## wherein the --R--NH-- moiety is ##STR2## R₁ ishydrogen, C₁ -C₄ alkyl, --COR₃, or --COXCO--; R₂ is hydrogen or--COXCO--; R₄ is hydrogen, C₁ -C₄ alkyl, amino, bromo, fluoro, chloro,or iodo; R₃ is hydrogen or C₁ -C₄ alkyl; X is a bond, C₁ -C₁₀ straightchain alkylene, C₂ -C₁₀ branched alkylene, C₂ -C₁₀ alkenylene, C₃ -C₁₀alkynylene, C₃ -C₆ cycloalkylene, phenylene, or hydroxysubstituted C₁-C₁₀ alkylene, and A is N or C--R_(4;) provided that one and only one ofR₁ and R₂ is --COXCO--.

Also provided by this invention are intermediates of the formula Q--OH,i.e., compounds of the formula ##STR3## wherein R₆ is hydrogen, C₁ -C₄alkyl, --COR₃ or --COXCOOH; R₇ is hydrogen or --COXCOOH; provided thatone and only one of R₆ and R₇ is --COXCOOH.

Compounds of formula III are useful as intermediates to the conjugatesof this invention, and also possess useful antitumor activity.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

This invention provides conjugates formed by the reaction of an acid offormula (III) in activated form with one or more amino groups of theimmunoglobulin molecule, for example, amino groups derived from lysineresidues.

Preferred conjugates are those wherein R₁ is hydrogen and --R--NH-- is a4-amino-2-oxo-1-pyrimidinyl radical. Preferred X groups are alkylenemoieties, particularly those containing 2-4 carbon atoms. Furtherpreferred conjugates are those wherein R₂ is hydrogen and R₁ is--COXCO--; and those wherein n is about 4-8.

Preferred intermediates are those of formula III wherein R₆ is--COXCOOH.

It is recognized that the difluoro ribosides of formulas I and III canexist either in the α- or β- form. This invention provides forconjugates having ribosides in either the racemic, or individual α- orpreferred β- forms.

The acids of formula III are prepared from the nucleosides of formula IV##STR4## wherein R₁ ' has the meanings of R₁ other than --COXCO--, bystandard techniques. Of course, R₁ ' must be hydrogen if the linkinggroup is to be placed on the 5'-methanol.

In general, the linking group is put in place by allowing an amine offormula IV to react with the mono activated diacid of the formulaZCOXCOOH (V). The moiety Z is a carboxy activating group such as thosewell known in the chemical art and in particular those used in peptidechemistry. Such groups are discussed, for example, in peptide synthesisby M. Bodanszky, et al., 2nd Edition, (1976) John Wiley & Sons, notablypages 85-136. Such values of Z, therefore, include an azide group(--N₃), a halo group, for example bromo and especially chloro, anacyloxy group of the formula R₅ COO where R₅ is an aliphatic or aromaticresidue such as C₁ -C₄ alkyl, an alkoxy group, preferably C₁ -C₃ alkoxyor an aryloxy group, a methanesulfonyloxy, tosyloxy, orbenzenesulfonyloxy group, an imidazolyl radical, or the residue of anN-acyl hydroxylamine derivative, for example where Z is succinimidoxy,phthalimidoxy or benzotriazolyloxy. Alternatively, a cyclic anhydride offormula VI may be employed in place of V. ##STR5## Such chemistry iswell known in the art, for example, for acylating alcohol moieties on amolecule, see, e.g., U.S. Pat. No. 4,596,676 and British PatentApplication No. 2,137,210. In general, compounds IV and V (or VI) arepreferably reacted in a non-reactive solvent such as ethers, for exampletetrahydrofuran, diethyl ether, dioxane, and the like, ketones, such asacetone, methyl ethyl ketone, and the like, hydrocarbons, such ashexane, cyclohexane, toluene, and the like, or chlorinated hydrocarbons,such as methylene chloride. Alternatively, or in addition to thesolvents, the reaction may be performed in or in the presence of anon-reactive acid scavenger such as pyridine, triethylamine, and thelike. It is advisable to use a substantial excess of the reagent offormula V or VI, particularly when the desired product has the linkinggroup on the 5'-methanol. Excess amounts in the range of 2×-6×,particularly about 4×, are desirable.

Reaction of the nucleoside of formula IV with the acylating reagentproduces a mixture of products. The amino group is most readilyacylated, but the 5'-methanol oxygen and the 3'-hydroxy oxygen are alsoreactive. Thus, not only monoacylated products but also bis- andtris-acylated products are formed. The various products are separated bychromatographic techniques which are shown in detail in the examplesbelow.

It is believed that the linking group on the 5'-methanol is the moststable of the three acylatable positions. Accordingly, the intermediateof formula III wherein R₆ is --COXCOOH can be isolated in quite pureform by cleaving any unwanted acyl groups from the amino nitrogen andthe 3'-hydroxy oxygen, as by simple exposure of an aqueous solution toheat. Such isolations are also shown below in the examples.

The thus formed compounds III can then be reacted with the chosenimmunoglobulin. The conjugation is accomplished by first activating theacid group of III with a Z functionality as previously described. Again,such methods of preparing acid halides, azides, activated esters, mixedanhydrides, and the like are well known in the art. Of particularimportance are activating groups such as succinimidoxy and acid halide,especially acid chloride, derivatives. These activated acids areprepared by standard techniques well known in the art and are then usedfor coupling with the immunoglobulin. The reaction of the immunoglobulinand the activated form of III is best employed in aqueous medium and ata temperature of about 5-25° C. and a pH of about 7-10. The processresults in the attachment by covalent linkage of one or more of themodified difluoronucleosides through the acid groups to form amides withthe free amino groups of the immunoglobulin molecule. The number ofresidues attached will depend on the concentration of the reactants, theduration of the reaction, and the particular immunoglobulin, but theaverage number is usually approximately 1-10.

In carrying out the conjugation of activated III and the immunoglobulin,a suitable solvent such as dimethylformamide is used as a vehicle tointroduce the activated acid into a buffered solution of immunoglobulinin, for example, 0.34M borate buffer at pH 8.6. The conjugate isisolated by gel filtration with a phosphate buffered saline at pH 7.4 asthe solvent. Alternatively, the conjugate can be stored in arefrigerator at 4° C. or frozen at, for example, -20° C.

The nucleosides of formula IV are known in the art. U.S. Pat. No.4,526,988, British Patent Application No. GB 2172287, and EPO PatentApplication No. 184,365 are expressly incorporated into this applicationas illustrative of the compounds which are represented by formula IV andas providing methods for their preparation. Reagents V and VI are eithercommercially available, known in the literature, or can be prepared bymethods known in the art.

The other component for the novel conjugates is an immunoglobulin and,of that class, preferably a monoclonal antibody (MoAb), which is agammaglobulin such as an IgG or an IgM. The preferred class ofimmunoglobulins are those which are reactive with antigens on thesurface of unwanted cells; i.e., are able to recognize antigens; haveantigen recognizing properties.

Techniques for the production of such immunoglobulins from the serum ofimmunized animals or by culturing hybridomas secreting monoclonalproducts are well known. The preferred type of antibody for use in theinvention is an immunoglobulin which is a gammaglobulin. IgG, IgA, IgE,and IgM subclasses are particularly preferred. Some representativeimmunoglobulins are as follows, mono- or polyclonal antibodies to

(i) human or animal tumor associated antigens;

(ii) human B- and T-cell antigens;

(iii) human Ia antigens;

(iv) viral, fungal and bacterial antigens; and

(v) cells involved in human inflammatory or allergic reactions.

Of the preferred antibodies to human or animal tumor associated antigensthere may be mentioned:

(i) Ig from goats or sheep immunized with carcinoembryonic antigen;

(ii) Ig from rabbit antiacute lymphoblastic leukemia serum;

(iii) Ig from various primate antisera raised against acutelymphoblastic leukemia, acute myleoblastic leukemia, chroniclymphoblastic leukemia and chronic granulocytic leukemia;

(iv) Ig from goats or sheep immunized with lung carcinoma cells, orcellular fractions;

(v) monoclonal Ig from mouse hybridomas secreting anti-human colorectalcarcinoma antibodies;

(vi) monoclonal Ig from mouse hybridomas secreting anti-human melanomaantibodies;

(vii) monoclonal Ig from mouse hybridomas that secrete antibodiesreacting with human leukemia cells;

(viii) monoclonal Ig from mouse hybridomas secreting antibodies reactingwith human neuroblastoma cells;

(ix) monoclonal Ig from mouse hybridomas secreting antibodies reactingwith human breast cancer antigens;

(x) monoclonal Ig from mouse hybridomas secreting antibodies reactingwith human ovarian carcinoma cells;

(xi) monoclonal Ig from mouse hybridomas secreting antibodies reactingwith human osteosarcoma cells, with human pancreatic carcinoma cells,with human prostatic carcinoma cells etc.;

(xii) monoclonal Ig from mouse hybridomas secreting antibodies toadenocarcinomas including lung, renal, breast and pancreas;

(xiii) monoclonal Ig from mouse hybridomas secreting antibodies reactingwith human squamous carcinoma cells;

(xiv) monoclonal Ig from human hybridomas (hybridomas which secreteantibodies to the human tumor-associated antigen including, but notlimited to, those monoclonals above);

(xv) any antibody or fragment thereof that contains carbohydrate ineither the light or heavy chain;

(xvi) Monoclonal Ig from rat, hamster, or other mammalian species notspecifically mentioned above, from hybridomas which secrete antibodiesto human tumor associated antigens including, but not limited to, thosementioned above.

As indicated above, the conjugate can also be made with immunoglobulinfragments Ig', referred to also as Fab, Fab', F(ab')₂ and IgM monomerderived from an antibody by, for example, proteolytic enzyme digestionor reductive alkylation. Useful conjugates are also made with chimericmonoclonal antibodies and bifunctional antibodies. Such materials andmethods of preparation are well known and it may be mentioned thatpreferred proteolytic enzymes are pepsin and papain. See generallyParham, J. Immunology, 131, 2895 (1983); Lamoyi et al., J. ImmunologicalMethods, 56, 235 (1983); Parham, id., 53, 133 (1982); and Matthew etal., id., 50, 239 (1982).

Specific MoAbs exist that are reactive against various tumors; suchimmunoglobulins which recognize antigens on the surface of, or otherwiseassociated with tumor cells, include but are not limited to thefollowing:

                  TABLE I                                                         ______________________________________                                        Tumor       MoAb         Reference                                            ______________________________________                                        Lung        KS1/4        N. m. Varki, et al.,                                                          Cancer Res. 44:681,                                                           1984                                                             534,F8;604A9 F. Cuttitta, et al.,                                                          in: G. L. Wright                                                              (ed) Monoclonal                                                               Antibodies and                                                                Cancer, Marcel                                                                Dekker, Inc., N.Y.,                                                           p. 161, 1984.                                        Squamous Lung                                                                             G1, LuCa2,   Kyoizumi et al.,                                     Cancer      LuCa3, LuCa4 Cancer Res., 45:3274,                                                         1985                                                             PF 1/D       Fernstein, et al.,                                                            Cancer Res., 46:2970,                                                         1986                                                             L1-KS, L2-KS Starling, et al.,                                                and L4-KS    Second Int'l. Conf.                                                           on Monoclonal Anti-                                                           body Immuno-                                                                  conjugates for                                                                Cancer, San Diego,                                                            Mar. 12-14, 1987                                     Small Cell Lung                                                                           TFS-2        Okabe et al., Cancer                                 Cancer                   Res. 45:1930, 1985                                   Colon       11.285.14    G. Rowland, et al.,                                              14.95.55     Cancer Immunol.                                                               Immunother., 19:1,                                                            1985.                                                            NS-3a-22,NS-10                                                                             Z. Steplewski, et al.,                                           NS-19-9,NS-33a                                                                             Cancer Res.,                                                     NS-52a,17-1A 41:2723, 1981.                                       Melanoma    9.2.27       T. F. Bumol and R. A.                                                         Reisfeld, Proc. Natl.                                                         Acad. Sci., (USA),                                                            79:1245, 1982                                                    p97          K. E. Hellstrom, et al.,                                                      Monoclonal Antibodies                                                         and Cancer, loc. cit.                                                         p. 31.                                                           R24          W. G. Dippold, et al.,                                                        Proc. Natl. Acad.                                                             Sci. (USA), 77:6114,                                                          1980.                                                Neuro-      P1 153/3     R. H. Kennet and                                     blastoma                 F. Gilbert, Science,                                                          203:1120, 1979.                                                  MIN 1        J. T. Kemshead in                                                             Monoclonal                                                                    Antibodies and                                                                Cancer, loc. cit.                                                             p. 49.                                                           UJ13A        Goldman et al., Pedi-                                                         atrics, 105:252, 1984.                               Glioma      BF7,GE2,CG12 N. de Tribolet, et al.,                                                       in Monoclonal                                                                 Antibodies and                                                                Cancer, loc. cit.                                                             p. 81.                                               Breast      B6.2,B72.3   D. Colcher, et al.,                                                           in Monoclonal                                                                 Antibodies and                                                                Cancer, loc. cit.                                                             p. 121.                                              Osteogenic  791T/48,     M. J. Embleton, ibid,                                Sarcoma     791T/36      p. 181                                               Leukemia    CALL 2       C. T. Teng, et al.,                                                           Lancet, 1:01, 1982.                                              anti-idiotype                                                                              R. A. Miller, et al.,                                                         N. Eng. J. Med.,                                                              306:517, 1982.                                       Ovary       OC 125       R. C. Bast, et al.,                                                           J. Clin. Invest.,                                                             68:1331, 1981.                                       Prostate    D83.21, P6.2,                                                                              J. J. Starling, et al.,                                          Turp-27      in Monoclonal                                                                 Antibodies and                                                                Cancer, loc. cit.                                                             p. 253.                                              Renal       A6H, D5D     P. H. Lange, et al.,                                                          Surgery, 98:143,                                                              1985.                                                ______________________________________                                    

Preferred conjugates are those prepared from monoclonal antibodies,especially those which recognize human cancer cells such asadenocarcinoma, squamous cell carcinoma, transitional cell carcinoma,melanoma, neuroblastoma, small cell carcinoma, leukemia, lymphoma, andsarcoma.

The following examples are illustrative of the conjugates inintermediates of this invention. These examples are illustrative onlyand are not intended to limit the invention in any way.

EXAMPLE 1 5'-O-(3-Carboxy-1-oxopropyl)-2'-deoxy-2',2'-difluorocytidine

Dry 2'-deoxy-2',2'-difluorocytidine (311 mg) was added to dry ethanoland brought to reflux. Succinic anhydride (2.5 g) was added in portionsover a half hour period and the mixture was refluxed an additional 1/2hour. The mixture was concentrated in vacuo and the resulting crudeproduct was placed on a silica gel column and eluted with a stepgradient of 5%, 20%, and 50% methanol in methylene chloride. The desiredproduct was isolated in the 50% methanol in methylene chloride wash andcontained 148 mg of material. This product was combined with productfrom a similar experiment providing a total of 253 mg of material. Thismaterial was taken up in 8 ml of 0.01M ammonium acetate solution. The pHwas adjusted with ammonium hydroxide to 8.6 and the solution placed on afreshly packed mono-Q anion exchange column (Pharmacia Inc., 800Centennial Avenue, Piscataway, N.J. 08854). Desired fractions werecombined, frozen, and lyophilized. The residue was dissolved indistilled water, frozen, and lyophilized several more times, thendissolved in a small amount of methanol and benzene, frozen, andlyophilized providing 60 mg of the desired title product. The proton NMRspectra was consistent with the structure of the desired product:

δ 7.68, doublet, 1H (C6,═CH--N); δ 6.24, triplet, 1H (C1', O--CRH--); δ6.13, doublet, 1H (C5, C═CH--); δ 4.78, singlet, 4 exchangable protons(--OH, --NH); δ 4.2-4.27, multiplet, 4 protons (sugar protons at C3',C4', C5'); δ 2.60 and 2.73, doublet of triplets, 4 protons (succinateprotons --CH₂ CH₂ --).

EXAMPLE 2 Conjugation of 5' -O-(3-carboxy-1-oxopropyl)-2'-deoxy-2',2'-difluorocytidine with 007B

A 25 mg sample of the product from Example 1 was dissolved in a smallvolume of benzene and methanol, frozen, and lyophilized a total of threetimes. The residue was dissolved in 1 ml of dry dimethylformamide and 4ml of freshly distilled tetrahydrofuran. The mixture was cooled to 4° C.and N-methyl morpholine (151 mcl) in tetrahydrofuran was added. After 10minutes, a 177 mcl sample of isobutyl chloroformate in tetrahydrofuranwas added. After stirring for 20 minutes at 4° C., 248 mcl ofN-hydroxysuccinimide were added. Stirring was continued for 20 minutesat 4° C. and for 3 hours at ambient temperature. The solution wasconcentrated in vacuo and kept under vacuum overnight.

The activated ester prepared in the preceding paragraph (31.7 mg) wasdissolved in 4.18 ml of dry dimethylformamide. Antibody 007B wasprepared by subcloning the cell line producing the published antibodyKSl/4 and selecting variant hybridoma clones which produce only therelevant IgG2a monoclonal antibody of KSl/4, and not the irrelevant IgGlprotein. Such clones were grown in cell culture by conventional methodsto produce 007B. Five hundred milligrams of 007B in pH 8.6 borate bufferwas placed in a round bottom flask and the solution of activated esteradded over a 10 minute period. The reaction was stirred in the dark for3 hours, adjusted to pH 7.4 with lN hydrochloric acid, and centrifugedfor 10 minutes at 2,000 rpm. The supernate was divided in half and eachportion placed on a Bio-gel P-6 column (Bio-Rad Laboratories, 32nd andGriffin Avenue, Richmond, California 94804) with phosphate bufferedsaline (PBS) at pH 7.4 as the eluant. The conjugate peaks were collectedin 3 fractions for each portion. Like fractions were combined from the 2runs. The combined second fractions were concentrated to 3 ml anddiluted with PBS to give a solution with a concentration of 25.44 mg/mlwith a total volume of 13.29 ml. The protein recovered was 338 mg andthe molar ratio of nucleoside to protein was 4:1. The product wassterilized and used for the following in vivo experiments.

EXAMPLE 3 5'-O-(3-Carboxy-1-oxopropyl)-2'-deoxy-2',2'-difluorocytidine

A 600 mg portion of dry 2'-deoxy-2',2'-difluorocytidine, hydrochloride,was added to 6 ml of dry pyridine, and 804 mg of succinic anhydride wasadded. The reaction mixture was stirred at ambient temperature for 1.5hours. The mixture was then concentrated to an oil under vacuum, and theoil was stored overnight in the freezer. It was then allowed to stand atambient temperature for 4 hours, and was chromatographed over a 500 mlQ-Sepharose column (Pharmacia Inc.). The oil was dissolved in 6 ml of0.lM ammonium acetate buffer, and was passed over the column, elutingwith a gradient buffer which changed from 0.lM to 1.0M ammonium acetate,at pH 8.0 at all times. Analytical chromatography of the mixture on asmall mono-Q column showed that bisacylated and tris-acylated productswere present, as well as mono-acylated product and a small amount ofstarting material. The product-containing fractions, amounting to about200 ml, were placed in a 37° C. bath, for 68 hours. At the end of thattime, the tris-acylated product had been decomposed and both mono- andbisacylated products were still present.

The product mixture was then separated by preparative chromatography ona mono-Q column, eluting with the same gradient buffer used above. Thefractions containing mono-acylated product were collected, pooled andfreeze-dried twice to obtain 508 mg of the desired intermediate product.The product was identified by nuclear magnetic resonance analysis, usingDzO as the solvent on a 300 mHz instrument. The characteristic featuresof the spectrum were δ 6.15, doublet, 1H (C5); 6.25, triplet, 1H (C1');7.7, doublet, 1H (C6).

EXAMPLE 4 5'-O-(3-Carboxy-1-oxopropyl)-2'-deoxy-2',2'-difluorocytidineN-(3-Carboxy-1-oxopropyl)-2'-deoxy-2',2'-difluorocytidin

Two hundred mg of 2'-deoxy-2',2'-difluorocytidine, hydrochloride, wasdissolved in 2 ml of pyridine, and then 134 mg of succinic anhydride wasadded. The mixture was stirred at ambient temperature for 1.25 hours,and was then concentrated under vacuum. The residue was dissolved indichloromethane/methanol, and the solution was absorbed on 1.5 g ofsilica gel, which was then slurried in dichloromethane and loaded on a 4g silica gel column. The column was eluted with a gradient solvent,starting with dichloromethane and ending with methanol. Theproduct-containing fractions were combined and freeze-dried overnight toobtain 255 mg of product mixture. It was dissolved in 3 ml of 0.01Mammonium acetate, and was chromatographed on a 22 ml mono-Q fast flowcolumn (Pharmacia Inc.), eluting with the ammonium acetate gradientbuffer which has previously been described. Evaporation of theproduct-containing fractions gave 81 mg of a mixture of themono-acylated products named in the heading.

The following nmr features of the two products were observed, usinganalysis as described in Example 3.

    ______________________________________                                                      N-acylated                                                                            O-acylated                                              ______________________________________                                        Triplet, 1H (C1')                                                                             δ 6.25                                                                            δ 6.25                                        Doublet, 1H (C5)                                                                              δ 7.4                                                                             δ 6.15                                        Doublet, 1H (C6)                                                                              δ 8.2                                                                             δ 7.7                                         ______________________________________                                    

EXAMPLE 5 Activation of5'-O-(3-carboxy-1-oxopropyl)-2'-deoxy-2',2'-difluorocytidine

A 23.2 mg portion of the intermediate compound of Example 1 was weighedinto a 50 ml flask, and was dissolved in a small amount of methanol.Then a few ml of benzene was added, and the solution was evaporatedunder vacuum. The dissolution and evaporation was repeated once, toassure that the compound was dry. It was then dissolved in 1.2 ml ofdimethylformamide, which had been stored in the presence of molecularsieve to dry it. A 14.7 mg portion of N-hydroxysuccinimide was added,followed by 14.5 mg of dicyclohexylcarbodiimide. The mixture was thenstirred overnight at ambient temperature, to obtain the succinimidoxyester of 5'-O-(3-carboxy-1-oxopropyl)-2'-deoxy-2',2'-difluorocytidine.

EXAMPLE 6 Conjugation with antibody 9.2.27

A 624 μ1 portion of the active ester solution from Example 5 was addedto a vial, and to it was added 100 mg of isolated antibody 9.2.27. Themixture was stirred at ambient temperature for a few hours, and was thenstored in the refrigerator for two days. The mixture was thenchromatographed over a G-50 size exclusion column (Pharmacia Inc.),eluting with PBS. The product-containing fractions were examined byultraviolet analysis, observing the maximum at 280 amd the minimum at251 λ. The ratio of drug to antibody was 3.7 moles of drug per mole ofantibody.

EXAMPLE 7 Conjugation with antibody 007B

An activated ester solution was prepared as described in Example 5, butin twice the concentration. A 1.4 ml portion of that solution was addedto 500 mg of isolated antibody 007B, and the mixture was stirred atambient temperature for four hours. It was then centrifuged, and thesolid pellet was loaded onto and chromatographed through a G-50 columnas described in Example 6 above. Two pools of product-containingfractions were obtained, and were analyzed by ultraviolet, as describedin Example 6. Analysis of the two pools showed that 8.9 and 10.2 molesof drug were conjugated to each mole of antibody, respectively.

Anti-Tumor Activity In the P3-UCLA Adenocarcinoma Model

The inhibition of the P3-UCLA adenocarcinoma in female nude mice wasdetermined by standard techniques. Inoculation of the mice was performedon day 0, compounds were dosed intravenously days 2, 4, and 8, andevaluation was made on days 14 or 21. The percent inhibition of tumorgrowth was determined employing controls receiving vehicle only. Groupsof 5 mice were used for each test and control group. The dose for theconjugate is expressed as the calculated mg/kg of parent nucleosideadministered. The results from these tests are summarized in Table II.

                  TABLE II                                                        ______________________________________                                        Inhibition of P3-UCLA Adenocarcinoma                                          in Nude Mice                                                                  Compound of                                                                              Dose         Percent Inhibition                                    Example    (mg/kg)*     14 days 21 days                                       ______________________________________                                        1          5.0          25%     37%                                           2          5.0          86%     84%                                                      2.5          63%     62%                                                      0.5          31%     27%                                           ______________________________________                                         *Dose administered intravenously on days 2, 4, and 8. Inoculation of cell     on day 0; evaluation on days 14 or 21. Dose for Example 2 calculated as       mg/kg of parent nucleoside.                                              

Additional tests of the same type were carried out, with observation ofthe animals, 14, 21 and 28 days after inoculation. The conjugate ofExample 2 was a pooled lot having an average conjugation ratio of 7.8.

    ______________________________________                                        Compound of Dose     Percent Inhibition                                       Example     (mg/kg)  14 days   21 days                                                                             28 days                                  ______________________________________                                        1           10       41        36    28                                                   2.5      28        18     2                                                   0.5      25         7    23                                       2           10       97        99    95                                                   5        97        98    94                                                   2.5      96        92    85                                                   1        48        38    32                                                   0.5      48        16     4                                       ______________________________________                                    

One of the five mice on the highest rate of Example 1 died, as did threeof the five mice on the highest rate of Example 2.

The above test results demonstrate the value of conjugating thenucleoside to a directing antibody. It is obvious from the data that theconjugated drug gives more than twice the inhibition of the tumor,compared to the parent nucleoside, even at lower, nontoxic doses.

In Vitro Affinity Testing

In vitro tests were carried out in an ELISA system to determine theaffinity of the antibody portion of the conjugated drug for an antigento which the unconjugated antibody is bound. The results are presentedas the fraction of the antibody present in the sample which binds to theantigen.

    ______________________________________                                        Conjugation Ratio                                                                            ELISA Result                                                   ______________________________________                                        Antibody 007B                                                                 8.9            0.43                                                           6.6            0.42                                                           2.5            0.55                                                           Antibody 9.2.27                                                               16.5           0.15                                                           4.9            0.26                                                           0              0.65                                                           ______________________________________                                    

The novel conjugates of the invention are useful in the treatment ofcancers and as such are preferably prepared for use in formulationssuitable for injection. Thus the invention includes a pharmaceuticalformulation, for example an injectable preparation comprising aconjugate of the invention together with a pharmaceutically-acceptablecarrier or diluent such as are well known in the art. The formulation ispreferably in unit dosage form, each dosage containing, for example,from 0.01 to 10 mg of the active ingredient (in terms of the nucleosidedrug moiety).

The novel conjugates are effective over a wide dosage range and dosagesper week, for example, for the treatment of adult humans suffering fromcancer will normally fall within the range of 0.01 to 10 mg/kg(nucleoside drug moiety), more usually in the range of from 0.03 to 9mg/kg. However it will be understood that the amount of conjugateactually administered will be determined by a physician in the light ofthe relevant circumstances, including the condition to be treated andthe chosen route of administration.

We claim:
 1. A conjugate of the formula (Q)_(n) --Im where Im is animmunoglobulin or immunoglobulin fragment, n is about 1-10, and Q is anacylated difluoronucleoside of the formula ##STR6## wherein the--R--NH-- moiety is ##STR7## R₁ is hydrogen, C₁ -C₄ alkyl, --COR₃, or--COXCO--; R₂ is hydrogen or --COXCO--; R₄ is hydrogen, C₁ -C₄ alkyl,amino, bromo, fluoro, chloro, or iodo; R₃ is hydrogen or C₁ -C₄ alkyl; Xis a bond, C₁ -C₁₀ straight chain alkylene, C₂ -C₁₀ branched alkylene,C₂ -C₁₀ alkenylene, C₃ -C₁₀ alkynylene, C₃ -C₆ cycloalkylene, phenylene,or hydroxysubstituted C₁ -C₁₀ alkylene, and A is N or C-R_(4;) providedthat one and only one of R₁ and R₂ is --COXCO--.
 2. A conjugate of claim1 wherein R₂ is hydrogen.
 3. A conjugate of claim 2 wherein --R--NH-- isa 4-amino-2-oxo-1-pyrimidinyl radical.
 4. A conjugate of claim 3 whereinX is C₂ -C₄ alkylene.
 5. A conjugate of claim 4 wherein Q is5'-O-(1,4-dioxobutyl)-2'-deoxy-2',2'-difluorocytidinyl.
 6. A conjugateaccording to claim 1 in which IM is an immunoglobulin which specificallybinds antigen.
 7. A conjugate according to claim 6 in which theimmunoglobulin is a monoclonal antibody.
 8. A conjugate according toclaim 7 in which the MoAb recognizes human cancer cells.
 9. A conjugateaccording to claim 8 in which the MoAb recognizes human cancer cellsselected from the group consisting of adenocarcinoma, squamous cellcarcinoma, transitional cell carcinoma, melanoma, neuroblastoma, smallcell carcinoma, leukemia, lymphoma, and sarcoma.
 10. A conjugateaccording to claim 9 wherein Q is5'-O-(1,4-dioxobutyl)-2'-deoxy-2',2'-difluorocytidinyl.
 11. A conjugateaccording to claim 9 wherein the MoAb is adapted for recognition ofhuman adenocarcinoma cells.
 12. A conjugate according to claim 10wherein the MoAb is adapted for recognition of human adenocarcinomacells.